This invention relates to the hydrolysis of oligosaccharides containing .alpha.-galactoside linkage in the presence of .alpha.-galactosidase and more particularly to a method for stabilizing .alpha.-galactosidase enzyme activity containing mycelia for subsequent use in the hydrolysis of oligosaccharides containing .alpha.-galactoside linkage.
The hydrolysis of oligosaccharides containing .alpha.-galactoside linkage, such as raffinose, stachyose, melibiose, galactobiose, verbascose, and the like, in the presence of .alpha.-galactosidase is an important commercial practice in some industries. For example, in the production of crystalline sugar from sugar beets, the naturally occurring trisaccharide raffinose is known to be present in sugar beet diffusion juice in varying quantities. Since raffinose forms insoluble calcium saccharates, it is precipitated together with sucrose in the conventional Steffen process, and tends to build up in Steffen molasses as sucrose is precipitated from the molasses solution. As with other impurities, the presence of raffinose in sucrose containing liquids detrimentally effects sucrose crystalization, such as by depressing the sucrose crystallization velocity and by resulting in the formation of cubic, flat or needle-like crystals at varying raffinose concentrations. In the past, it has been a common commercial practice to discard, or use merely as a by-product, molasses having a raffinose buildup of over about 5% by weight on dry substance without attempting to recover additional sucrose from the molasses. Such practices have resulted in the loss of substantial quantities of potentially recoverable sucrose.
To overcome the foregoing problems, it has previously been suggested to treat raffinose containing beet molasses with .alpha.-galactosidase to hydrolize the raffinose into D-galactose and sucrose, thereby permitting the recovery of additional sucrose from the molasses solution. For example, U.S. Pat. No. 3,647,625 of Suzuki et al relates to such a process wherein .alpha.-galactosidase is formed in mycelia of Mortierella vinacea var. raffinose-utilizer (ATCC No. 20034) to obtain mycelia substantially free of invertase activity. The foregoing .alpha.-galactosidase containing mycelia is commercially provided in pellet form to allow for continuous enzymatic treatment in a sucrose production process. Although this process has been successful in reducing the raffinose content of beet molasses and thereby increasing recoverable sucrose yields, it has been found that enzyme losses are relatively high due to actual physical loss of the mycelia, loss of .alpha.-galactosidase from the mycelia and/or inactivation of the .alpha.-galactosidase. Losses of each type substantially effect the realized enzyme activity level in a continuous raffinose hydrolysis process and the economic desirability of conducting such a process.
In addition, it has previously been known that glucose isomerase activity within whole Streptomyces olivaceus (NRRL 3583) bacterial cells can be stabilized by treating the whole bacterial cells with glutaraldehyde. See, for example, U.S. Pat. No. 3,779,869 of Zienty, which relates to such a process. However, there is no disclosure or suggestion in the Zienty patent that glutaraldehyde treatment might be effective for stabilization of anything other than glucose isomerase activity in whole bacterial cells.
In accordance with the present invention, it has been found that the .alpha.-galactosidase activity level in the hydrolysis of oligosaccharides containing .alpha.-galactoside linkage can be stabilized by treating mycelial bound .alpha.-galactosidase with about 5 to about 25 percent by weight glutaraldehyde based upon the dry weight of the mycelia. The treatment method is preferably performed by suspending .alpha.-galactosidase activity containing mycelia in an aqueous medium, adding an active site protection agent to the aqueous medium, adding the glutaraldehyde to the aqueous medium, maintaining the pH of the aqueous medium within the range of about 6.5 to about 8.5, mixing the aqueous medium for a period of at least about 0.25 hours, separating the mycelia from the aqueous medium and then washing the mycelia to remove excess glutaraldehyde. The treated .alpha.-galactosidase activity containing mycelia may then be used in a conventional manner in the hydrolysis of oligosaccharides containing .alpha.-galactoside linkage.